Heat treatment to decrease chilling injury in tomato fruit. Effects on lipids, pericarp lesions and fungal growth

Tomato fruits are sensitive to low temperature and develop chilling injury, while at nonchilling temperatures they ripen rapidly. Previously, a hot-air treatment was found to reduce the sensitivity of the fruit to low temperatures. In the present study hot air was compared to hot water and their effects on reducing chilling injury and fungal decay were investigated. Tomatoes ( Lycopersicon esculentum cv. Daniella) at the breaker stage were subjected to hot air, 48 h at 38°C, or various hot water dips, 30 min at 40°C or 2 min at 46, 48 or 50°C, before holding at 2°C. The unheated tomatoes developed chilling injury and fungal infections at 2°C, but not at 12°C. All the heat treatments reduced chilling injury and decay in tomatoes held for 3 weeks at 2°C. The outer pericarp tissue of heated tomatoes had higher phospholipid and lower sterol contents than unheated tomatoes. Heated tomatoes also had less saturated fatty acids than unheated tomatoes held at 2°C, but not at 12°C. Scanning electron micrograph observations showed that all the fruits had microcracks in their surface, but the unheated chilled tomatoes had also fungal growth in the cracks, while those of the heated tomato fruit did not. In the areas of chilling injury collapsed cells were present under the peel and could also support pathogen development. It is suggested that the heat treatment institutes a response to high temperature stress in the fruit tissue that leads to strengthened membranes. This prevents the loss of function and cell collapse which was found in the chilling-injured areas of affected fruit.     

Lamellar dispersion and phase separation of chloroplast membrane lipids by negative staining electron microscopy

Aqueous dispersions of lipids isolated from spinach chloroplast membranes were studied by electron microscopy after negative staining with phosphotungstic acid. Influence of low temperature (5°C for 24 h) was also investigated. It was observed that when contacted with water, these lipids, as such, formed multilamellar structures. Upon sonication, these multilamellar structures gave rise to a clear suspension of unilamellar vesicles varying in size (diameter) between 250 and 750 Å. When samples of sonicated unilamellar vesicles were stored at 5°C for 24 h or more, they revealed a variety of lipid aggregates including liposomes, cylindrical rods (about 100 Å wide and up to 3600 Å long), and spherical micellar structures (100–200 Å in diameter)—thus indicating phase separation of lipids.     

Microsomal membrane lipids from pea shoots and Ranunculus sceleratus petioles

Microsomal membranes, relatively free of chloroplasts and mitochondria, were prepared from etiolated pea stems and Ranunculus sceleratus petioles by differential centrifugation. Relative quantitative analyses were made of the phospholipid, glycolipid, sterol and fatty acid contents.     

Thermodynamic interpretation of effects of alcohols on membrane lipid fluidity

The effect of a series of amphiphilic compounds, the first eight n-aliphatic alcohols, on the fluidity of rat enterocyte brush border was determined by ESR using 5-doxyl stearic acid as a lipid spin probe. Packing order variations are compared to the relative hydrophobic effect of the alcohols. The concentrations, [Ci]5 of each alcohol that decrease the membrane 2T' value by 5%, vary by a factor of 1500 from methanol to octanol. From [Ci]5, the membrane concentrations Cm and the variation of free energy delta F degree due to the incorporation of the alcohols in the lipids, were calculated. These calculations were performed taking into account the respective volumes of the aqueous phase and the membrane lipids. Cm is of the order of 0.18 mol/kg for the odd chain length alcohols and of 0.27 mol/kg for the even alcohols. The value of delta F degree in cal/mol -CH2- is -687 cal on average for the eight alcohols. This work shows that for all the alcohols, the concentrations at equilibrium in the membrane and in the aqueous phase are respectively in agreement with Meyer and Overton's theory and with the gradient of free energy which constitutes the most general index of interaction of lipophilic substances with membranes.     

Prevention of imbibitional injury in low vigor soybean embryonic axes by osmotic control of water uptake

Deterioration of soybean [ Glycine max (L.) Merr. cv. Essex] seeds during accelerated aging at 41°C and 100% relative humidity predisposes the embryonic axis to injury during the initial period of imbibition. This injury was prevented or greatly reduced in severity when excised axes were imbibed on blotters containing 30% polyethylene glycol which slowed the rate of water uptake and when axes were pre-equilibrated to a high moisture level. Rates of water uptake by "high"(no treatment) and "low vigor"(accelerated aged) excised axes were identical. However, high vigor axes tolerated rapid water uptake during early imbition, whereas low vigor axes did not. Leakage of electrolytes during early imbibition was nearly six times greater in low than in high vigor axes. Polyethylene glycol significantly reduced the leakage of electrolytes from both low and high vigor axes. The data are in agreement with the hypothesis that seed deterioration in soybeans involves membrane changes which may predispose embryonic tissues to injury during imbibition. Reduction of the rate of water uptake during the initial period of imbibition would allow extra time for membrane repair or rearrangement, thus permitting the tissues to develop in a more orderly manner. The data indicate that deterioration in soybean seeds involves, at least in part, a decrease in ability of seed axes to tolerate rapid water uptake at the start of imbibition and that this weakness may be compensated by osmotic control of water uptake.     

Various aspects of inhibition of photosynthesis under light/chilling stress: “Photoinhibition at chilling temperatures” versus “chilling damage in the light”

At chilling temperatures, plants suffer damage to photosynthesis. The sites and the mechanisms involved in this damage differ under different chilling conditions. The current status of our understanding of this damage is reviewed, and how chilling temperatures affect photosynthesis is discussed with emphasis on the role of light and the phase separation of membrane lipids. Recipient of the Botanical Society Award for Young Scientist, 1996     

Archaeal lipids and their biotechnological applications

The lipids of Archaea, based on glycerol isopranoid ethers, can be used taxonomically to distinguish between phenotypic subgroups of the domain to delineate them clearly from all other organisms. This review is a general survey of the structural features of archaeal lipids and how they relate to survival in the harsh environments in which the Archaea live. The molecular organization of archaeal lipids in monolayers, artificial black membranes and vesicles and the unique properties and possible biotechnological applications of liposomes of the lipids are presented. The results with these liposomes are compared with similar data obtained with synthetic compounds which mimic the structure of archaeal lipids. Studies on computer simulation are also reported.A. Gambacorta is with the istituto per la Chimica di Molecole di interesse Biologico, CNR via Toiano 6, 80072 Arco Felice, Napoli, Italy; A Gliozzi is with the Dipartimento di Fisica, Università di Genova, via Dodecanneso 33, 16146 Genova, Italy. M. De Rosa is with the Istituto di Biochimica delle Macromolecole. Seconda Università di Napoli, via Costantinopoli 16, 80132 Napoli, Italy.     

Germination and ion leakage are linked with phase transitions: of membrane lipids during imbibition of Typha latifolia pollen

In previous studies on the causes of imbibitional leakage in dry polien we have presented data which suggest that the leakage is due to a gel to liquid crystalline phase transition in membrane phospholipids during the rehydration event. In the present study we greatly extend and confirm those results. A supplemented phase diagram for the hydration dependent transition temperature of membrane phospholipids in pollen is presented. In pollen containing > 0.05 g H₂O g⁻¹ dry weight at the time of imbibition, this phase diagram for the phospholipids precisely predicts the conditions for rehydration under which germination is maximal and leakage is minimal. However, in extremely dry pollen, containing < 0.05 g H₂O g⁻¹ dry weight the predictive value of the phase diagram for phospholipids in the pollen is not in agreement with data for germination and leakage. Thus, an alternative explanation must be sought for leakage in these circumstances. We examined the available evidence and suggest here that a modified form of the non-bilayer phase hypothesis proposed by Simon (1974) may apply in the specialized case of extremely dry cells.     

烟草种子成熟过程中膜脂的变化规律研究

该研究采用脂类组学方法,系统地研究了烟草种子成熟过程中膜脂含量及组成比例的变化规律。结果表明:(1)构成叶绿体和类囊体膜的重要脂类质体膜脂的含量及其在总膜脂中的组成比例,在种子成熟的整个过程中保持下降趋势;而构成细胞膜的重要脂类质外体膜脂含量在种子成熟前期则下降显著,在授粉21 d后基本保持不变。(2)总膜脂含量的变化规律与质体膜脂类似,但在授粉后第29天后含量却达到稳定状态。(3)因油脂在种子成熟过程中不断积累,且化学结构与膜脂相似,质体膜脂含量的降低可能与种子成熟过程中种子对油脂累积的持续需求以及对叶绿体及类囊体的需求降低有关。(4)质外体膜脂含量在授粉21 d后基本保持不变的原因,可能是由于脂质外体膜脂是细胞膜组成的主要膜脂,细胞膜在种子成熟以及成熟种子萌发过程中均发挥重要作用,因此质外体膜脂只在种子成熟的前期有部分转化为油脂。     

Ascorbate-glutathione cycle of mitochondria in osmoprimed soybean cotyledons in response to imbibitional chilling injury

Osmopriming treatment of chilling-sensitive soybean (Glycine max L. cv. Zhonghuang-22) seeds for 72 h at 25 °C with polyethylene glycol (PEG8000) solution at −1.5 MPa strongly improves chilling resistance. The aim of the present work was to investigate whether the beneficial effect of osmopriming is associated with restoration of the ascorbate-glutathione (ASC-GSH) cycle of mitochondria in soybean seeds. Compared with the control, both H₂O₂ and malondialdehyde (MDA) contents in mitochondria of osmoprimed seeds decreased after chilling treatment, and these changes were associated with increased activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and l-galactono-γ-lactone dehydrogenase (GLDH). However, the activity of dehydroascorbate reductase (DHAR) showed no obvious change during osmopriming treatment. Increased ASC and GSH contents accompanied prolonged osmopriming, and the reduced/oxidized ratios of ASC and GSH increased differently during osmopriming. These results indicate that osmopriming treatment enhances activity of the ASC-GSH cycle of mitochondria, which raises the chilling tolerance in soybean seeds and protects against H₂O₂ that is generated in mitochondria during imbibition at low temperature.     

Response to chilling of tomato mesophyll protoplasts

Freshly isolated protoplasts from tomato leaves show two completely different responses to a chilling treatment of 12 h at 7° C prior to culture at 29° C, depending on the presence or absence of glucose in the medium. In the culture medium with glucose as osmoticum, where the rate of cell divisions under optimal culture conditions is relatively high (about 20% plating efficiency), protoplasts were drastically injured by the chilling procedure and died. In the medium with mannitol as the osmoticum instead of glucose, where the plating efficiency even under optimal conditions is rather low (about 8%), protoplasts withstand the chilling procedure. More-over, after the chilling treatment when the protoplasts were transferred to the optimal culture temperature of 29° C, the plating efficiency was raised to about 20%, which is the same level as in the glucose-containing medium without chilling. This effect was not observed when the medium in which the protoplasts were suspended during the chilling period was replaced with fresh medium. This suggests that under these conditions tomato protoplasts produce and excrete a factor in the cold that improves the vitality of the cells or stimulates cell division. The possible relationship between chilling sensitivity of tomato protoplasts and their ability to divide will be discussed.     

Alleviating effects of exogenous Gamma-aminobutiric acid on tomato seedling under chilling stress

Low temperature during germination and early seedling growth is one of the most significant limiting factors in the productivity of plants. Tomato seedling germination is sensitive to chilling stress. Gamma-aminobutyric acid (GABA), as a non-protein amino acid, involved in various stress tolerances in plants. In this study, 5-day old tomato seedlings were exposed to chilling stress (2 ± 0.05 °C for 48 h) and then the effects of 0, 100, 250, 500 and 750 μmolL⁻¹ concentrations of GABA on electrolyte leakage, proline and malondialdehyde (MDA) content were investigated. The resultS showed that the antioxidant enzyme activity, electrolyte leakage, MDA and proline content were significantly reduced by GABA treatments. However under chilling stress seedlings treated with GABA exhibited significantly higher sugar and proline contents as compared to un-treated seedlings. These results suggest that GABA treatment protects tomato seedlings from chilling stress by enhancing some antioxidant enzymes activity and reducing MDA content which results in maintaining membrane integrity.     

Rapid determination of rice seed vigour by spontaneous chemiluminescence and singlet oxygen generation during early imbibition

Using a highly sensitive single photon counter, a spontaneous chemiluminescence (CL) study on rice (Oryza sativa L.) seeds stored in different years was carried out. We first observed that the degree of ageing in rice seeds was related to the intensity of spontaneous CL during early imbibition (0–30 min). Rice seeds stored for a shorter time had a stronger intensity of CL in early imbibition. The germination rate of rice seeds showed an obvious positive correlation with the intensity of spontaneous CL. Singlet oxygen (¹O₂) in rice seeds during early imbibition was investigated by a CL method using a cypridina luciferin analogue, 2‐methyl‐6‐(p‐methoxyphenyl)‐3,7‐dihydroimidazo [1,2α] pyrazin‐3‐one (MCLA), as a selective CL probe. Additional experimental evidence for the formation of ¹O₂ came from the quenching effect of sodium azide (NaN₃) on MCLA‐mediated rice seeds' CL. Analysis based on the experimental results demonstrated that spontaneous CL in rice seeds during early imbibition was mainly contributed by singlet oxygen (¹O₂). Copyright © 2002 John Wiley & Sons, Ltd.     

Polar lipids from the radiation resistant bacterium Deinococcus radiodurans: structural investigations on glucosaminyl and N-acetyl glucosaminyl lipids

Deinococcus radiodurans, although a gram-positive bacterium, has a complex cell wall with multiple layers and associates to this structural particularity, a quite unusual lipid composition for gram-positive bacteria. The conventional phospholipids (phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl glycerol...) are absent. Among the nine polar lipids detected in the R1 Anderson strain, three are glycolipids only one is a phospholipid, the other ones are glycophospholipids. One of the latter compounds contains one free amino group. Analysis by aminoacid autoanalyser enables to identify glucosamine in one glycolipid and in two glycophospholipids. Sugar analysis by gas-liquid chromatography after acid methanolysis and trifluoroacetylation, reveals the occurrence of N-acetyl glucosaminyl residues in one glycolipid and in one phospholipid. The following identification for the two lipids of D. radiodurans is proposed: phosphatidyl glucosaminyl glycerol and phosphatidyl N-acetyl glucosaminyl glycerol.     

Degradation of leaf polar lipids during chilling and post-chilling rewarming of Zea mays genotypes reflects differences in their response to chilling stress. The role of galactolipase

Degradation of leaf polar lipids [monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG)] and chlorophyll (Chl) were studied in four Zea mays genotypes differing in chilling susceptibility following dark chilling and post-chilling rewarming at original growth conditions. Assessment of visual chilling injury symptoms during post-chilling rewarming differentiated maize inbred lines into chiling-sensitive (CS) CM7 and Co151 lines and chillingtolerant (CT) S215 and EP1 lines. Severity of chilling injury in CS and CT inbreeds were correlated with the extent of Chl and polar lipids degradation. Chilling for either 4 or 6 days followed by 4 days of rewarming caused more extensive degradation of total polar lipids content in CS than in CT lines. MGDG decreased mostly during chilling whereas DGDG dropped during rewarming only. Chl content was not affected during chilling but its large decrease, greater in CS than in CT lines, was observed upon rewarming. Extent of polar lipids breakdown in CS and CT inbreeds during chilling and post-chilling rewarming is correlated with galactolipase activity in chloroplasts (Kaniuga et al., 1998) and visual assessment of chilling injury. In view of the data it is likely that contribution of galactolipase activity induced during low-temperature stress of CS plants is an important factor responsible for thylakoid lipid degradation and development of chilling injury as postulated previously (Kaniuga 1997). It is suggested that genetically engineered reduction of galactolipase activity or elimination of the factors(s) involved in induction/stimulation of its activity during chilling might increase tolerance of CS species to chilling stress.     

Principles of membrane stability and phase behavior under extreme conditions

Biological membranes consist of a complex assortment of lipids and proteins. The arrangement of the components, particularly in regard to their lateral disposition in the plane of the membrane under physiological conditions, is dependent on the phase behavior of the different membrane lipids and the way that this behavior is modified by interaction with other membrane components and electrolytes in the aqueous medium. Irreversible phase separation of components within the membrane may result from exposure to extreme environmental conditions including temperature, pressure, or electrolyte concentration. The principles underlying the phase-mixing behavior of model membrane systems can be used to provide useful information about the factors that determine the stability of biomembranes under physiological and non-physiological conditions. These data are reviewed and used to predict events that take place when membranes are exposed to environmental stress.     

Effects of paclobutrazol and chilling temperatures on lipids, antioxidants and ATPase activity of plasma membrane isolated from green bell pepper fruits

The effects of paclobutrazol treatment on plasma membrane lipid composition and ATPase activity of bell pepper fruit ( Capsicum annuum ) subjected to chilling temperatures were assessed. Application of the growth regulator paclobutrazol affected plant growth and fruit morphology. The plants were more compact and the fruits were less elongated than control fruits. There was about 60% more plasma membrane on a fresh weight basis from treated fruits. At harvest there was no difference in sterol to phospholipid ratio, or in phospholipid fatty acid composition of control compared with paclobutrazol treated fruit. However, plasma membrane ATPase acitivity of treated fruit was two times higher than that of control fruit. After storage at chilling temperature (2°C), the control fruit developed more chilling iniury, and had greater weight loss and a higher rate of K⁺ leakage than paclobulrazol treated fruit. Plasma membrane phospholipid content decreased and saturation of phospholipid fatty acids was higher than in control fruit. These two changes were largely absent in plasma membrane from treated fruit. At harvest antioxidant levels in the plasma membrane of paclobutrazol treated peppers were higher than in those of controls and changed little during storage, whereas levels in control fruit plasma membrane decreased 66%. ATPase activity increased and then decreased in control fruit held at low temperature, whereas in treated fruit activity was constant. The protective effect of paclobutrazol against chilling injury of pepper fruit may result from a combination of its effect on fruit morphology, and protection of the lipids against oxidative stress.     

The role of membrane lipids in the induction of macrophage apoptosis by microparticles

Microparticles are membrane-derived vesicles that are released from cells during activation or cell death. These particles can serve as mediators of intercellular cross-talk and induce a variety of cellular responses. Previous studies have shown that macrophages undergo apoptosis after phagocytosing microparticles. Here, we have addressed the hypothesis that microparticles trigger this process via lipid pathways. In these experiments, microparticles induced apoptosis in primary macrophage cells or cell lines (RAW 264.7 or U937) with up to a 5-fold increase. Preincubation of macrophages with phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)BP) reduced the microparticle-induced apoptosis in a dose-dependent manner. PtdIns(3,5)BP is a specific inhibitor of the acid sphingomyelinase and thus can block the generation of pro-apoptotic ceramides. Similarly, the pre-incubation of macrophages with PtdIns(3,5)BP prevented microparticle-induced upregulation of caspase 8, which is a major target molecule of ceramide action in the apoptosis pathway. PtdIns(3,5)BP, however, had no effect on the spontaneous rate of apoptosis. To evaluate further signaling pathways induced by microparticles, the extracellular signal regulated kinase (ERK-) 1 was investigated. This kinase plays a role in activating phospholipases A2 which cleaves membrane phospholipids into arachidonic acid; microparticles have been suggested to be a preferred substrate for phospholipases A2. As shown in our experiments, microparticles strongly increased the amount of phosphorylated ERK1/2 in RAW 264.7 macrophages in a time-dependent manner, peaking 15 min after co-incubation. Addition of PD98059, a specific inhibitor of ERK1, prevented the increase in apoptosis of RAW 264.7 macrophages. Together, these data suggest that microparticles perturb lipid homeostasis of macrophages and thereby induce apoptosis. These results emphasize the importance of biolipids in the cellular cross-talk of immune cells. Based on the fact that in clinical situations with excessive cell death such as malignancies, autoimmune diseases and following chemotherapies high levels of circulating microparticles might modulate phagocytosing cells, a suppression of the immune response might occur due to loss of macrophages.     

Modulation of membrane fluidity by catalytic hydrogenation affects the chilling susceptibility of the blue-green alga, Anacystis nidulans

All the changes, i.e. the phase separation temperature of thylakoid lipids, shift in the chilling induced increase of K⁺ permeability and decline in photosynthetic O₂-production, respectively, brought about by temperature acclimation in Anacystis nidulans, can be accomplished by homogeneous catalytic hydrogenation of the fatty acids, as well, using a new water-soluble Pd(II) complex, hitherto unknown in biological applications. Since the thermo-adaptation replaced by proper hydrogenation conducted under isothermal condition results in a similar modification of chilling susceptibility, it afforts direct evidence that chilling response is mediated by changing the degree of fatty acid unsaturation in Anacystis nidulans.     

The effect of liposomes on thermotropic membrane phase transitions of bovine spermatozoa and oocytes: implications for reducing chilling sensitivity

We have examined the effects of combinations between egg-phosphatidylcholine (EPC) or dipalmitoylphosphatidylcholine (DPPC) liposomes with either bovine spermatozoa or oocytes on cellular chilling sensitivity, lipid phase transition temperature (T(m)), and the ability of the oocytes to develop to the blastocyst stage. Spermatozoa and oocytes were exposed to EPC and DPPC liposomes at various temperatures (spermatozoa: 4, 12, 16, and 25 degrees C; oocytes: 4, 16, and 32 degrees C). The membrane integrity of the spermatozoa-control group decreased significantly following exposure to 16 or 12 degrees C, compared to ambient temperature (25 degrees C). In contrast, the EPC-sperm group had a greater resistance to chilling at each temperature and showed a decline in membrane integrity only at the lowest temperatures investigated. However, the DPPC-sperm group was injured significantly at all temperatures tested. Similar to the sperm, oocytes from the control group that were exposed to 16 degrees C were injured more severely than oocytes that were electrofused with EPC or DPPC liposomes. The membrane integrity of the oocytes at 16 degrees C that were electrofused with either EPC or DPPC liposomes was approximately the same as the control group held at 32 degrees C (normalized to 100%), compared to 46% in the control group at 16 degrees C (P<0.01). The transition temperatures of the sperm and oocyte membranes revealed different T(m) for the different liposome treatments. All groups had a significantly higher cleavage rate, as well as increased blastocyst formation when oocytes were exposed to temperatures above or below their T(m). We suggest that the T(m) of spermatozoa or oocytes can be changed by spontaneous association or electrofusion of liposomes with cellular membranes and, consequently, the chilling sensitivity can be altered. The resulting possibility is that embryo development after cryopreservation could be improved with such a method.